Molecular characterization and population dynamics of lactic acid bacteria during the fermentation of sorghum

Ting is a cooked fermented sorghum food that is popular amongst southern Africans for its sour taste and unique flavour. However, major challenges are associated with large-scale production and marketing of this spontaneously fermented food due to inconsistent microbiological and sensory quality. The use of starter cultures may circumvent these limitations. Prior to engaging starter cultures, detailed knowledge of the microbial diversity and dynamics during fermentation is important. Therefore, the aim of this study was to investigate microbial diversity and dynamics during sorghum fermentations, and to clarify the role of starter cultures regarding the microbiological safety and consumer acceptance of sensory characteristics of fermented ting. A culture-independent approach, based on the use of PCR-denaturing gradient gel electrophoresis (DGGE), revealed that Lactococcus lactis, Lactobacillus curvatus, Weissella cibaria and some Enterobacteriaceae were predominant at the end of spontaneous sorghum fermentations. Culture-dependent methods indicated that Lb. fermentum, Lb. plantarum, Lb. rhamnosus, E. faecalis, E. mundtii, W. cibaria and L. lactis were predominant at the end of fermentation. These results not only indicated the predominant bacteria during sorghum fermentation, but also indicated that a combined approach is required to reveal microbial diversity and dynamics during spontaneous sorghum fermentations. Based on the above results, L. lactis, Lb. fermentum, Lb. plantarum and Lb. rhamnosus were evaluated as starter cultures for production of ting. All the starter cultures were able to ferment sorghum, but the lowest pH and highest lactic acid was produced in naturally fermented sorghum inoculated with L. lactis. This fermentation showed an increase in the number of lactic acid bacteria and yeasts, whilst pathogen counts decreased. Ting from this fermented gruel, in contrast to naturally fermented sorghum, had sensory properties preferred by panelists. The results indicated that the use of L. lactis in starter cultures may result in ting with consistent and acceptable attributes.Thesis (PhD)--University of Pretoria, 2011.Microbiology and Plant Pathologyunrestricte

Prevalence of Vibrio cholerae in rivers of Mpumalanga province, South Africa as revealed by polyphasic characterization

Cholera is a life-threatening diarrhoeal disease, which mainly affects inhabitants of developing countries due to poor socio-economic conditions and lack of access to potable water and sanitation. Toxigenic Vibrio cholerae are the aetiological agents of cholera. These bacteria are autochthonous to aquatic environments, hence water plays a central role both in the epidemiology and transmission of cholera. The aim of this study was to determine the prevalence of V. cholerae from 32 sites of major rivers in Mpumalanga province of South Africa using a polyphasic approach. Water samples (594) collected over for 4 months were cultured on thiosulphate-citrate-bile salt-sucrose agar, and oxidase positive (88) isolates were subjected to biochemical tests and duplex polymerase chain reaction targeting the outer membrane protein (ompW) and cholera toxin (ctxAB) genes. All ompW PCR positive V. cholerae isolates were subjected to rfbO1 PCR. Fifteen isolates from Crocodile, Komati and Gutshwa rivers were assigned to V. cholerae by both biochemical tests and PCR, of which no isolates were positive for ctxAB and rfbO1 genes. The polyphasic approach was effective at revealing non-O1 and non-toxigenic V. cholerae in some rivers. Such information is important for raising awareness regarding the presence of V. cholerae so that precautionary measures are taken on time.Key words: Vibrio cholerae, ompW gene, ctx gene, rfbO 1, surveillance

Polyphasic taxonomic characterization of lactic acid bacteria isolated from spontaneous sorghum fermentations used to produce ting, a traditional South African food

Ting, an indigenous cooked fermented food made from sorghum flour, is consumed extensively in South Africa. Due to the spontaneous nature of the sorghum fermentation considerable variations in the sensory and microbial quality of the end-product may occur, thus hampering large-scale production of this food. The use of starter cultures purified from the fermented sorghum may be an alternative approach to obtain ting of consistent quality. The aim of this study was therefore to identify the lactic acid bacteria (LAB) associated with ting fermentation using a polyphasic approach. Phenotypic characterization and sequence analysis of the genes encoding the 16S subunit of the ribosomal RNA (rrs) and phenylalanyl tRNA synthase (pheS) were used. The results of these analyses showed that ting fermentation involved at least three different species of LAB, i.e. Lactobacillus fermentum, L. plantarum and L. rhamnosus. To our knowledge, this is the first report of polyphasic taxonomic characterization of LAB from this food. This research forms an essential first step towards the development of relevant starter cultures to produce ting of consistent quality

Investigation of multidrug-resistant fatal colisepticaemia in weanling pigs

Escherichia coli is usually a benign commensal of the gut microflora. However, when E. coli acquires virulence genes it can multiply rapidly and cause disease through colonisation of the intestinal mucosa. Escherichia coli can become a significant pathogen in young pigs. We report an investigation of fatal colisepticaemia in weanling pigs from emerging farms where piglets and weaners were diarrhoeic and the mortality rate ranged between 15% and 70% in each litter. Faecal and tissue samples were processed for histopathology, bacteriology and molecular biology (multiplex and monoplex polymerase chain reaction) and we recovered enteroaggregative multidrug-resistant E. coli producing EAST-1 enterotoxin. An association between poor housing conditions and the observed cases was established and future management programmes were recommended to reduce the impact of such pathogens. Enteroaggregative E. coli is becoming a major problem in the pig industry. It therefore becomes necessary to establish the full impact of E. coli on the South African pig industry and to determine the geographic extent of the problem.http://www.ojvr.orgam2015ab201

Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa

Abstract Background Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. Results In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter’s box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. Conclusions PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes

Effect of usage / non-usage of antibiotics on virulence profiles of Escherichia coli in pig production

Pathogenic Escherichia coli pathogens are responsible for acute profuse diarrhoea in growing pig with resultant high morbidity and mortality. Enterotoxigenic E. coli (ETEC) encoding STa, STb, EAST1 and LT enterotoxins and Shiga toxin E. coli (STEC) encoding Stx2e occur most commonly in pigs. This study investigated the prevalence of ETEC and VTEC virulence genes in two groups of growing pigs (five piglets per group) kept under routine farm management practices. One group was administered antibiotics and the other group received no antibiotics. A total of 241 E.coli strains were isolated in piglets from both groups between 0 and 70 days of age. Virulence genes were detected by PCR in 24.8% (18.2 - 32.7) of the antibiotic group isolates and 43.5% (34.5 - 52.9) of the non-antibiotic group with a significant difference (P = 0.002). The proportions of the virulence genes STa, STb, EAST1 and Stx2e were 18.1% (8.61 - 34.39), 0% (0.0 - 10.43), 78.7% (62.25 - 89.32) and 3% (0.53 - 15.32) in the antibiotic group respectively, and 14.8% (7.40 - 27.68), 8.5% (3.36 - 19.93), 85.1% (72.32 - 92.59) and 12.7% (5.98 - 25.17) in the non-antibiotic group respectively. AIDA1 was the most dominant non-fimbrial adhesion factor while F6 was the only fimbrial factor detected. Twelve pathotypes were identified, with pathotype EAST1 being the most prevalent. The study showed that usage/non-usage of antibiotics in growing pigs does not prevent occurrence of disease causing virulence genes and other factors may be involved.Poster presented at the University of Pretoria, Faculty of Veterinary Science Faculty Day, September 07, 2017, Pretoria, South Africa.ab201

Antimicrobial usage in pig production : effects on Escherichia coli virulence profiles and antimicrobial resistance

Antimicrobials (AM) are used for growth promotion and therapy in pig production. Its misuse has led to the development of resistant organisms. We evaluated Escherichia coli virulence genes, and compared phenotypic–genotypic antimicrobial resistance (AMR) patterns of faecal E. coli from pigs receiving routine farm treatment without antimicrobial agents against pigs treated routinely with AM over 70 days. Recovered E. coli were tested for AMR using disk diffusion and polymerase chain reaction. Virulence genes were detected in 24.8% of isolates from antimicrobial group and 43.5% from non-antimicrobial group (p = 0.002). The proportion of virulence genes heat-stable enterotoxins a & b (STa, STb), enteroaggregative heat stable enterotoxin 1 [EAST1] and Shiga toxin type 2e [Stx2e]) were 18.1%, 0.0%, 78.7% and 3.0% for antimicrobial group and 14.8%, 8.5%, 85.1% and 12.7% for non-antimicrobial groups, respectively. Resistance to oxytetracycline was most common (p = 0.03) in samples collected between days 10 and 21. Resistance shifted to amoxicillin on days 56–70, and trimethoprim resistance was observed throughout. Seventeen phenotypic AMR combinations were observed and eight were multidrug resistant. At least one tetracycline resistance gene was found in 63.9% of the isolates. tet (A) (23.3%) was most common in the antimicrobial group, whereas tet (B) (43.5%) was prevalent in the nonantimicrobial group. Usage or non-usage of antimicrobial agents in growing pigs does not preclude virulence genes development and other complex factors may be involved as previously described. Heavily used AM correspond to the degree of resistance and tetracycline resistance genes were detected during the growth phase.The National Research Foundation Incentive Funding for Rated Researchers (IPRR) and the University of Pretoria Postgraduate Bursary.http://www.ojvr.orgpm2020Production Animal StudiesVeterinary Tropical Disease

Prevalence, risk factors and molecular characteristics of Shiga toxin-producing Escherichia coli in beef abattoirs in Gauteng, South Africa

Please read abstract in the article.Red Meat Research and Development South Africa (NAS2015-0116) and the University of Pretoria.https://www.elsevier.com/locate/foodconthj2022Production Animal Studie

Some South African Rubiaceae tree leaf extracts have antimycobacterial activity against pathogenic and non-pathogenic Mycobacterium species

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic M. smegmatis, M. aurum and M. bovis BCG using a two-fold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. MIC values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied and Oxyanthus speciosus had the most promising selectivity index values.The University of Pretoria Institutional Research Theme for Animal and Zoonotic Diseases (IRT-AZD), National Research Foundation (NRF, Grant No 81010) and Medical Research Council (MRC).http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-15732016-07-30hb201

Shiga toxin–producing Escherichia coli contamination of raw beef and beef-based ready-to-eat products at retail outlets in Pretoria, South Africa

A cross-sectional study was conducted to determine the prevalence of and factors associated with Shiga toxin–producing Escherichia coli (STEC) in raw beef and ready-to-eat (RTE) beef products sold in 31 retail outlets in Pretoria, South Africa, and nearby areas. A total of 463 beef and RTE samples were screened for four STEC virulence genes (stx1, stx2, eaeA, and hlyA) and seven O-serogroups (O113, O157, O26, O91, O145, O111, and O103) with a multiplex PCR assay. The total aerobic plate count (TAPC) per gram was also determined. A total of 38 STEC isolates were recovered and characterized by conventional PCR assay and serotyping. The overall prevalence of STEC in the beef and RTE samples tested was 16.4% (76 of 463 samples; 95% confidence interval, 13 to 20%). The prevalence of STEC differed significantly by product type (P < 0.0001), with the highest prevalence (35%) detected in boerewors (spicy sausage). The STEC prevalences in minced beef, brisket, RTE cold beef, and biltong were 18, 13, 9, and 5%, respectively. The most frequently detected stx gene was stx2 (13%), and STEC serogroups from recovered isolates were detected at the following prevalences: O2, 15%; O8, 12%; O13, 15%; O20, 8%; O24, 3%; O39, 3%; O41, 8%; O71, 3%; O76, 3%; O150, 12%; and O174, 3%. A high proportion (77%) of the samples had TAPCs that exceeded the South African microbiological standards for meat export (5.0 log CFU/g). The prevalence of O157 STEC (16%) and the diversity of non-O157 STEC serogroups found in five common beef-based products from retail outlets in South Africa suggest exposure of raw beef and beef products to multiple contamination sources during carcass processing and/or cutting and handling at retail outlets. These data provide direct estimates of the potential health risk to consumers from undercooked contaminated products and indicate the need to improve sanitary practices during slaughter and processing of beef and beef-based RTE products. A risk-based surveillance system for STEC may be needed.Red Meat Research and Development, South Africahttps://www.foodprotection.org/publications/journal-of-food-protection2021-02-17hj2020Production Animal Studie